Chromatographically pure galactosylceramide I3-sulfate (cerebroside sulfate (CBS)) containing palmitic acid or D-2-hydroxypalmitic acid has been prepared by the acylation of galactosylsphingosine I3-sulfate obtained from the saponification of bovine brain sulfatides. Optically pure D-2-hydroxypalmitic acid was obtained by adapting literature methods for the synthesis of the racemic acid and its resolution. The thermotropic behavior of the two synthetic CBSs were compared to each other and to the corresponding components in natural CBS, obtained by fractionation of bovine brain sulfatides, in order to determine the contribution of the hydroxy fatty acid to intermolecular hydrogen bonding between molecules of the lipid. The gel to liquid crystalline phase transition temperature (Tc) of the hydroxy fatty acid (HFA) synthetic form is 53.2 degrees C, 3 degrees higher than that of the non-hydroxy fatty acid (NFA) form at low concentrations of Na+ or K+. A similar difference was found for the HFA and NFA forms of natural CBS. The enthalpy of the NFA synthetic form is 8.5 kcal/mol, about 30% greater than that of the HFA form. The difference in Tc between the NFA and HFA forms is abolished as the Na+ or K+ concentration increases but the difference in enthalpy persists. Increasing cation concentration, over the range 0.01-2 M, increases Tc more than for an acidic phospholipid, phosphatidylglycerol, probably due to increased intermolecular hydrogen bonding as the charged sulfate is shielded. K+ causes a 3-4 degrees C greater increase in Tc relative to that produced by Na+ while K+ and Na+ have similar effects on phosphatidylglycerol.